1. Technical Field
The present invention relates to a method of detecting a pathogenic microorganism in real-time, using a modified flow-type surface plasmon resonance (SPR) biosensor and, more particularly, to a method of detecting a pathogenic microorganism in real-time, using a modified flow-type SPR biosensor, which separates an immune reaction from surface binding reaction in a conventional flow-type SPR sensor, thereby increasing the limit of real-time detection of a pathogenic microorganism by the SPR sensor.
2. Discussion of Related Art
As industry has been developed, people have been exposed to a lot of environmental pollutions and have faced serious problems of health and sanitation. Among various environmental pollutions, the drink water pollution frequently causes infection accidents by pathogenic microorganisms.
Compared to those in the past, the infection accidents of waterborne epidemic have been significantly reduced. However, it is true that no country in the world eradicates the waterborne epidemic. The waterborne epidemic by the water pollution causes very serious troubles because it has harmful effects for a short period of time. While the water pollution by chemical materials causes a chronic disease which is long-lasting and recurrent, the water pollution by waterborne pathogen causes an acute disease to develop symptoms and results for a short period and to be likely to be spread by secondary infection. So, prevention is very important in the infection caused by waterborne pathogen. In this regard, if the presence of pathogenic microorganism in water is detected, waterborne diseases are expected to be prevented in advance.
Many methods of detecting and monitoring a pathogenic microorganism have been developed based on diverse application principles, such as the immunofluorescence assay, specifically, the enzyme-linked immunosorbent assay (ELISA), and the gene detection assay by amplifying the polymerase chain reaction (PCR). Some of products by the aforementioned methods have been commercialized. However, among the methods of detecting a pathogenic microorganism, the aforementioned assays have a relatively high detecting ability but have drawbacks in that a sample needs to be pretreated and it takes a long time for an immune reaction. Accordingly, a new sensor for detecting a pathogenic microorganism needs to be developed.
To solve the drawbacks of conventional sensors for detecting a pathogenic microorganism, many researchers have been actively developing sensors for detecting pathogenic microorganisms, using a surface plasmon resonance (SPR) system which is capable of detecting a pathogenic microorganism in real-time and has the function of a label-free sensor.
Specifically, the technique of an SPR sensor is widely used as a biosensor and/or biochip measurement method, by signal changes occurring when biological materials, such as proteins, are bound with the surface of the sensor (Literature: Nice, E. C. and Catimel, B., BioEssay, 1999, 21, 339-352). The surface plasmon is a quantized oscillation of a free electron which is propagated along a conductive surface, such as a metal surface. The surface plasmon is excited by light which passes a dielectric medium, such as a prism, and then which is incident upon a metal thin film at an angle being same as or greater than a critical angle of the dielectric medium. Then, the surface plasmon generates resonance at a predetermined angle. An angle of incidence at which the SPR is generated, that is, an angle of resonance, is sensitive to a change in the index of refraction of a material being close to the metal thin film. The SPR sensor with the above-described feature is used for quantitative analysis, qualitative analysis and measurement of the thickness of a sample, by the change in the index of refraction of a material (that is, the sample) being close to the metal thin film. Compared to conventional immunoassays, the SPR sensor has several merits as follows:
First, in terms of specificity, a conventional immunoassay needs to use a specific label material (a luminous substance or a fluorescent substance) or a secondary antibody for measurement, thereby changing the activity or original properties of the sample to be measured. However, since an SPR sensor does not require for any label of a specific material or any pretreatment of the sample, it measures the sample while maintaining the original properties of the sample.
Second, in terms of sensitivity, a conventional immunoassay has the limit of detection which is generally from several tens of ng unit to several ug unit as for proteins. However, a SPR sensor is capable of detection to several pg unit, thereby having high sensitivity.
Third, in terms of time for measurement, a conventional immunoassay obtains a measurement result after several hours starting from the pretreatment of the sample, if short, or several days, if long. However, a SPR sensor does not need any special pretreatment of the sample, so that it has the promptness to obtain a measurement result within several minutes to several tens of minutes.
Last, in terms of simplicity of analysis procedure, a general immunoassay needs highly skilled personnel and includes a complicated pretreatment process of the sample for experimental procedure reasons. However, a SPR sensor obtains a measurement result by only injecting the sample on the surface of a sensor chip, so that it has the simplified analysis procedure, compared to the conventional immunoassay.
However, unlike an inter-immune reaction between proteins, an immune reaction to detect a pathogenic microorganism is slow in an immune reaction speed. In the immune reaction, detection signals are weakly measured due to the signal amplification characteristics of the SPR. Accordingly, it is difficult to use the SPR sensor to detect a pathogenic microorganism as a sensor for detecting a pathogenic microorganism, by applying a flow-type method like a conventional SPR sensor for detecting proteins.
Most of the conventional SPR sensors for detecting a pathogenic microorganism apply a batch-type method not a flow-type method. When using the batch-type sensors, it is difficult to realize the promptness of detection, which is one of the merits of the aforementioned SPR sensor.
Accordingly, a method of detecting a pathogenic microorganism in real-time, using a modified flow-type SPR sensor system is provided, in order that the conventional SPR sensor for detecting a pathogenic microorganism is used, with the promptness of detection, as a SPR sensor for detecting a pathogenic microorganism in real-time.